The mechanism of regulation of fibroblast growth factor-2 (fgf-2) and vascular endothelial growth factor-a (vegf-a) dependent angiogenesis by junctional adhesion molecule-a (jam-a).

摘要

Angiogenesis is the process of growth of new blood vessels from pre-existing ones. Angiogenesis is regulated by growth factors and integrin mediated signaling. The two well known growth factors that control angiogenesis are fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor-A (VEGF-A). Signaling induced by growth factors and mediated via integrins cause endothelial cells to migrate and form new blood vessels. Junctional Adhesion Molecule-A (JAM-A) is a tight junction protein that regulates angiogenesis by modulating FGF-2 induced pathway. However the FGF-2-induced signaling pathway dependent on JAM-A is currently unknown. Therefore I aimed to study the mechanism by which JAM-A regulated FGF-2 induced angiogenesis and if JAM-A played a role in VEGF-A induced angiogenesis Here I elucidate the different residues and motifs of JAM-A involved in the process of FGF-2-induced cell migration. Using primary endothelial cells isolated from WT and Jam-A deficient mice, I have shown FGF-2-induced cell migration, adhesion, ERK1/2 activation and Rap1 activation is dependent on Jam-A in endothelial cells. I show that JAM-A associates with postsynaptic density 95/disc-large/zonaoccludens (PDZ) domain containing protein Zona occludens-1 (ZO-1) in unstimulated cells and upon stimulation associates with Afadin (AF-6). Cells expressing JAM-A mutants with PDZ binding motif deletion show reduced FGF-2- induced Rap1 activation, ERK1/2 activation as well as cell migration suggesting that the PDZ binding motif of JAM-A plays an important role in FGF-2-induced pathway. I further show JAM-A to be serine 284 phosphorylated and associated with integrin beta3 in unstimulated conditions. However upon FGF-2 stimulation JAM-A gets tyrosine phosphorylated, which is accompanied by dephosphorylation of JAM-A phosphoserine 284 and dissociation from activated integrin beta3. The tyrosine phosphorylation of JAM-A seems to be specifically important as I show that FGF-2-induced cell migration is also dependent on the tyrosine residue of JAM-A. Upon FGF-2 stimulation, JAM-A is tyrosine phosphorylated and associates with C-src tyrosine kinase (CSK). This association between JAM-A and CSK is found to be through the SH2 domain of CSK. Our results suggest that FGF-2-induced signaling in endothelial cells is dependent on the tyrosine residue and the PDZ binding motif of JAM-A by interacting with other signaling proteins. Although Jam-A deficient mice showed ablated FGF-2 induced angiogenesis, I report spontaneous corneal opacity associated with inflammation, angiogenesis and the presence of myofibroblasts in Jam-A deficient mice. Since wounds and/or corneal infections cause corneal opacities, I tested the role of Jam-A in wound-induced inflammation, angiogenesis and scarring by subjecting Jam-A deficient mice to deep penetrating corneal wounding. Analysis of these wounds demonstrated increased inflammation, angiogenesis, and increased number of myofibroblasts thereby indicating that Jam-A regulates the wound-healing response by negatively controlling wound-induced inflammation, angiogenesis and scarring in the cornea. These effects were not due to inflammation alone, since the inflammation-induced wound-healing response in Jam-A deficient mice was similar to wild type mice. In order to determine the molecular mechanism associated with the observed aberrant corneal wound healing in Jam-A deficient mice, I assessed the expression of the components of VEGF-A/vascular endothelial growth factor receptor- 2(VEGFR-2) signaling pathway. Interestingly, I observed increased levels of VEGF-A mRNA in Jam-A deficient eyes. I also observed nuclear localization of phosphorylated SMAD3 (pSMAD3) indicative of transforming growth factor beta (TGF-beta) pathway activation in the Jam-A deficient eyes. Furthermore the increased wound-induced corneal inflammation, angiogenesis, and scarring in Jam-A deficient mice was attenuated by treatment with DC101, an anti-vascular endothelial growth factor receptor-2 (VEGFR-2) antibody. Our results suggest that in the absence of Jam-A, the VEGF-A/VEGFR-2 pathway is upregulated, thereby augmenting wound induced corneal inflammation, angiogenesis, and myofibroblast accumulation leading to scarring. Taken together these data suggest that JAM-A positively regulates angiogenesis by modulating FGF-2 induced angiogenesis and regulates VEGF-A induced angiogenesis by negatively regulating VEGF-A expression.